Vaccine composition comprising il-12 adjuvant encapsulated in controlled-release microsphere

ABSTRACT

Disclosed is a vaccine composition including a pathogenic antigen and IL-12 encapsulated in controlled release microspheres. Also, the present invention discloses a method of enhancing the adjuvant effect of IL-12 by employing an IL-12 adjuvant encapsulated in controlled release microspheres. IL-12, used as an adjuvant for a co-administered vaccine antigen in the vaccine composition, is released in vivo for a prolonged period of time by being encapsulated in controlled release microspheres, thereby maximizing its adjuvant effect.

TECHNICAL FIELD

The present invention relates to a vaccine composition comprising apathogenic antigen and an IL-12 adjuvant encapsulated in controlledrelease microspheres. Also, the present invention is concerned with amethod of enhancing an adjuvant effect of IL-12 by employing an IL-12adjuvant encapsulated in controlled release microspheres.

BACKGROUND ART

The immune system uses various defense mechanisms for attackingpathogens, but not all of these mechanisms are activated afterimmunization. Protective immunity induced by vaccination is dependent onthe capacity of a vaccine to elicit an appropriate immune response toresist, control or eliminate a pathogen. Depending on the pathogen, thisrequires a cellular (cell-mediated) or humoral immune response, which isdetermined by the nature of the T cells that was activated afterimmunization. For example, many bacterial, protozoal and intracellularparasitic and viral infections appear to require a strong cellularimmune response for protection, while other pathogens, such ashelminths, primarily respond to a humoral response.

Adjuvants are substances that enhance immune responses toward foreignantigens including pathogenic organisms. Suitable adjuvants includesubstances that do not serve as antigens in hosts but enhance immunityby increasing the activity of cells of the immune system. Adjuvants havebeen reported to function in various ways, including by increasing thesurface area of an antigen, prolonging the retention of an antigen inthe body to allow time for the lymphoid system to access the antigen,slowing the release of an antigen, targeting an antigen to macrophages,activating macrophages, and eliciting non-specific activation of thecells of the immune system (H. S. Warren et al., Annu. Rev. Immunol.,4:369 (1986).

Typical adjutants include water and oil emulsions, for example, Freund'sadjuvant, and chemical compounds such as aluminum hydroxide or alum. Atpresent, alum is the only practically used adjuvant. When alum isadministered to the body in a form being bound to a protein, it is ableto induce sustained release of the protein. However, in this case, alumitself coverts antigen-specific immune responses to Th2-type immuneresponses. Since, typically, Th1 responses, rather than Th2, responsesare effective in inducing preventive immunity to pathogenic antigens,alum has limited application.

Current studies have been directed to the development of a method ofdelivering an antigen together with a cytokine involved in the inductionof immune responses to achieve an immune-enhancing effect. Adjuvantsbelonging to this category include interleukins such as cytokines, forexample, IL-1 or IL-12. In addition, adjuvants that do not followmechanisms of interleukins but belong to this category includeinterferons, especially gamma-interferon and alpha-interferon, tumornecrosis factor (TNF) and granulocyte macrophage colony stimulatingfactor (GM-CSF).

When injected into the body in protein forms, the aforementionedcytokines have problems of being easily removed from the body due totheir short half-lives and instability. According to previous studies,the persistence of cytokines is essential in effectively inducingantigen-specific immune responses (Sanjay Gurunathan et al., NatureMedicine 1998, 4:1409-1415). Thus, there is an urgent need for thedevelopment of methods capable of overcoming the problems and thusallowing effective vaccine development.

DISCLOSURE OF THE INVENTION

Leading to the present invention, the intensive and thorough researchinto the effect of IL-12 on vaccination when used as an adjuvant in avaccine composition in the form of being encapsulated in microspherescapable of achieving slow and sustained release of IL-12 in vivo,conducted by the present inventors, resulted in the finding that IL-12encapsulated in microspheres remarkably increases immune responses to avaccine for a prolonged period of time even in small amounts incomparison with a non-encapsulated protein form or a DNA form of IL-12.

Therefore, the present invention aims to maximize the adjuvant effect ofIL-12 by employing IL-12 encapsulated in controlled release microspheresas an adjuvant in a vaccine composition.

The present invention relates to a vaccine composition for enhancing theadjuvant effect of IL-12 comprising a pathogenic antigen and an IL-12adjuvant encapsulated in controlled release microspheres.

In addition, the present invention relates to a method of enhancing theadjuvant effect of IL-12, which is based on employing, as an adjuvant,an IL-12 adjuvant encapsulated in controlled release microspheres in avaccine composition comprising a pathogenic antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIGS. 1 a to 1 f are graphs showing the antibody responses in micesubcutaneously immunized with a hepatitis B virus surface antigen,HBsAg, and rIL-12-encapsulating microspheres, wherein the titers oftotal serum IgG, IgG1, and IgG2a antibodies were measured by an anti-SELISA, and each group was immunized with the following composition:

-   -   Group 1: HBsAg (0.5 μg)    -   Group 2: HBsAg (0.5 μg)+mock microspheres    -   Group 3: HBsAg (0.5 μg)+mock microspheres+rIL-12 (0.1 μg)    -   Group 4: HBsAg (0.5 μg)+rIL-12-encapsulating microspheres (0.1        μg);

FIGS. 2 a to 2 c are graphs showing the adjuvant effect ofrIL-12-encapsulating microspheres in mice immunized with various amountsof an antigen, wherein the adjuvant effect of the microspheres wasanalyzed by anti-S ELISA, and each group was immunized with thefollowing composition:

-   -   Group 1: HBsAg (0.1 μg)    -   Group 2: HBsAg (0.1 μg)+rIL-12-encapsulating microspheres (0.1        μg)    -   Group 3: HBsAg (0.5 μg)    -   Group 4: HBsAg (0.5 μg)+rIL-12 (0.1 μg)    -   Group 5: HBsAg (0.5 μg)+rIL-12-encapsulating microspheres (0.1        μg)    -   Group 6: HBsAg (2.5 μg)    -   Group 7: HBsAg (2.5 μg)+rIL-12-encapsulating microspheres (0.1        μg);

FIGS. 3 a to 3 c are graphs showing the results of an IFN-γ ELISPOTassay of CD8⁺ T cells stimulated with an HBV S-specific CTL epitope(IPQSLDSWWTSL), which were isolated from mice subcutaneously immunizedwith HBsAg and rIL-12-encapsulating microspheres, wherein each group inFIG. 3 a was immunized with the following composition:

-   -   Group 1: HBsAg (0.5 μg)    -   Group 2: HBsAg (0.5 μg)+mock microspheres    -   Group 3: HBsAg (0.5 μg)+mock microspheres+rIL-12 (0.1 μg.)    -   Group 4: HBsAg (0.5 μg)+rIL-12-encapsulating microsphere (0.1        μg), and

each group in FIGS. 3 b and 3 c was immunized with the followingcomposition:

-   -   Group 1: HBsAg (0.5 μg)    -   Group 2: HBsAg (0.5 μg)+rIL-12 (0.1 μg)    -   Group 3: HBsAg (0.5 μg)+rIL-12-encapsulating microspheres (0.1        μg)    -   Group 4: HBsAg (2.5 μg)    -   Group 5: HBsAg (2.5 μg)+rIL-12-encapsulating microspheres (0.1        μg);

FIGS. 4 a and 4 b show the results of intracellular staining using FACSto determine the adjuvant effect of rIL-12-encapsulating microspheres,wherein mice were immunized intranasally twice at intervals of 2 weekswith M2/82-90 peptide, known as a respiratory syncytial virus-specificCTL epitope, and rIL-12-encapsulating microspheres, and each group wasimmunized with the following composition:

-   -   Group 1: M2/82-90 (20 μg)+mock microspheres    -   Group 2: M2/82-90 (20 μg)+rIL-12-encapsulating microspheres (0.1        μg);

FIGS. 5 a and 5 b are graphs showing the antibody responses of miceimmunized with HBsAg and rIL-12-encapsulating microspheres to compareIL-12 DNA and IL-12 protein encapsulated in microspheres for adjuvanteffects, wherein the titers of total serum IgG, IgG1, and IgG2aantibodies were measured by an anti-S ELISA, IL-12 DNA wasintramuscularly administered, HBsAg and IL-12 protein-encapsulatingmicrospheres were subcutaneously administered, and each group wasimmunized with the following composition:

-   -   Group 1: HBsAg (0.5 μg)    -   Group 2: HBsAg (0.5 μg)+IL-12 DNA vaccine (10 μg)    -   Group 3: HBsAg (0.5 μg)+rIL-12-encapsulating microsphere (0.1        μg);

FIG. 6 is a graph showing the antibody responses of mice intranasallyimmunized with an influenza virus surface antigen, influenza HA, andrIL-12-encapsulating microspheres, wherein the titers of total serumIgG, IgG1, and IgG2a antibodies were measured by an anti-S ELISA, andeach group was immunized with the following composition:

-   -   Group 1: HA (3 μg)    -   Group 2: HA (3 μg)+rIL-12 (0.1 μg)    -   Group 3: HA (3 μg)+rIL-12-encapsulating microspheres (0.1 μg)    -   Group 4: HA (3 μg)+rIL-12-encapsulating microspheres (0.02 μg);

FIGS. 7 a to 7 d are graphs showing the results of intracellularstaining using FACs of CD8₊ T cells stimulated with an HA-specific CTLepitope, which were isolated from the mouse lung tissue at five daysafter influenza infection. The mice were intranasally immunized with aninfluenza virus surface antigen, HA protein, and rIL-12-encapsulatingmicrospheres, and each mice was challenged with lethal doses ofinfluenza virus at 9 weeks after last immunization. Each group wasimmunized with the following composition:

-   -   Group 1: HA (3 μg)    -   Group 2: HA (3 μg)+rIL-12 (0.1 μg)    -   Group 3: HA (3 μg)+rIL-12-encapsulating microspheres (0.1 μg)    -   Group 4: HA (3 μg) +rIL-12-encapsulating microspheres (0.02 μg);        and

FIG. 8 is a graph showing the survival rate of mice which wereintranasally challenged with an influenza virus surface antigen, HAprotein, and rIL-12-encapsulating microspheres and were infected withlethal doses of influenza virus by an intranasal route, wherein eachgroup was immunized with the following composition:

-   -   Group 1: HA (3 μg)    -   Group 2: HA (3 μg)+rIL-12 (0.1 μg)    -   Group 3: HA (3 μg)+rIL-12-encapsulating microspheres (0.1 μg)    -   Group 4: HA (3 μg)+rIL-12-encapsulating microspheres (0.02 μg).

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention provides a vaccine composition forenhancing the adjuvant effect of IL-12 comprising a pathogenic antigenand an IL-12 adjuvant encapsulated in controlled release microspheres.

The term “pathogenic antigen”, as used herein, refers to an antigen thatis derived from a pathogenic microorganism to which a host induces animmune response. The pathogenic microorganism may include anintracellular parasite, such as a virus, bacterium or protozoan, and anextracellular parasite, such as a helminth or bacterium.

The pathogenic antigen from a pathogenic microorganism includes proteinsor fragments thereof (e.g., protein degradation products), peptides(e.g., synthetic peptides, polypeptides), glycoproteins, carbohydrates(e.g., polysaccharides), lipids, glycolipids, hapten conjugates, wholeorganisms (killed or attenuated organisms) or portions thereof, toxinsand toxoids.

In addition, the pathogenic antigen may be a DNA sequence encoding anantigen from a pathogenic microorganism. This DNA sequence, togetherwith a suitable promoter sequence, may be directly used as an antigenadministered with a cytokine adjuvant. Alternatively, the DNA sequencemay be introduced into other vaccine strains of the pathogenicmicroorganism, and, upon expression in vivo, may provide an antigen.

The pathogenic antigen may be obtained or induced from a variety ofpathogens or organisms. For example, the pathogenic antigen may beobtained or induced from bacteria (e.g., Salmonella dublin, Borreliaburgdorferi, Bacillus, treptococcus, Bordetella, Listeria, Bacillusanthracis, Streptococcus pneumoniae, Neiseria meningiditis, H.influenza, etc.); viruses (e.g., hepatitis B virus, hepatitis C virus,acute respiratory virus, measles virus, poliovirus, humanimmunodeficiency virus, influenza virus, parainfluenza virus,respiratory syncytial virus, herpes simplex virus, Ebola virus,lymphocytic choriomeningitis virus, murine retrovirus, Rabies virus,Smallpox virus, adenovirus, Varicella-zoster virus, enterovirus,rotavirus, yellow fever virus, etc.); mycobacteria (e.g., Mycobacteriumtuberculosis, etc.); parasites (e.g., Leishmania, Schistosomes,Tranpanosomes, toxoplasma, pneumocystis, etc.); and fungi (e.g.,Histoplasma, Candida, Cryptococcus, Coccidiodes, Aspergillus, etc.), butthe present invention is not limited to these examples.

Preferably, the pathogenic antigen contained in the vaccine compositionof the present invention may be obtained or induced from viruses. Forexample, the pathogenic antigen may be derived from a broad range ofviruses including hepatitis viruses, acute respiratory virus, measlesvirus, poliovirus, human immunodeficiency virus, influenza virus,parainfluenza virus and respiratory syncytial virus.

In particular, in the case of viruses causing chronic diseases or havinghigh mutation rates, such as hepatitis B virus, hepatitis C virus, humanimmunodeficiency virus and influenza virus, Th1-type T cell immuneresponses are known to be more important in inducing preventive immunityor eliminating viruses than antibody immune responses, and IL-12 isknown to be essential for eliciting such immune responses. Also, in thecase of bacteria such as Mycobacterium tuberculosis, elevation of T cellimmune responses by IL-12 is known to be critical in inducing preventiveimmunity. Thus, the pathogenic antigen contained in the vaccinecomposition of the present invention is preferably derived fromhepatitis B virus, hepatitis C virus, human immunodeficiency virus,influenza virus or Mycobacterium.

The pathogenic antigen contained in the vaccine composition of thepresent invention may be obtained using techniques known in the art. Forexample, the antigen may be directly isolated (purified) from apathogen, induced using a chemical synthetic method, or using arecombinant DNA method. Also, the antigen may be obtained fromcommercially available products. The antigen useful in the presentinvention includes one or more B and/or T cell epitopes (e.g., T helpercell or cytotoxic T cell epitopes), and may be easily determined bythose skilled in the art.

Preferably, the vaccine composition of the present invention may includea pathogenic antigen in a protein or peptide form. Preferably, a proteinor peptide form of the pathogenic antigen may be directly isolated,chemically synthesized or prepared by a recombinant DNA technique, andmore preferably by the recombinant DNA technique.

If desired, the pathogenic antigen contained in the vaccine compositionof the present invention, as described above, may be contained in adispersion system to achieve its sustained release, which is selectedfrom the group consisting of macromolecular complexes, nanocapsules,microspheres, beads, oil-in-water emulsions, micelles, mixed micelles,liposomes and resealed erythrocytes.

Interleukin-12 (IL-12), contained in the vaccine composition of thepresent invention as an adjuvant, is known to be a major element inenhancing the efficacy of a vaccine when cellular immunity is required.

IL-12 is secreted by antigen presenting cells (APC) includingmacrophages and monocytes after appropriate stimulation, and functionsto modulate various immune responses in vivo. In detail, IL-12 has abroad range of biological activities including the differentiation of Thelper 1 (Th1) cells and natural killer (NK) cells, the regulation ofproduction of various cytokines, the enhancement of immune responsesmediated by Th1 cells, the differentiation of CD8⁺ T cells and theproliferation of hematopoietic cells (Hsieh, C. S., et al., Science,260:547-549, 1993). In particular, IL-12 plays a critical role inregulating immune responses by improving the hydrolysis capacity of CTLcells (cytotoxic T lymphocytes) and NK cells (Robertson, M. J., and J.Ritz., Oncologist, 1:88-97, 1999; Trinchieri, G., Annu. Rev. Immunol.,13:251-276, 1995). According to other reports, synthesis of biologicallyactive IL-12 decreases by about five times in AIDS patients (Chehimi, J.et al., J. Exp. Med., 179:1361-1366, 1994), and immunity againstmycobacteria greatly decreases in IL-12 receptor-deficient patients (deJong R. et al., Science, 280:1435-1438, 1998). Since IL-12, by virtue ofthese biological activities, can induce potent in vivo immune responsesagainst viruses, bacteria or various cancers in early stages, it isincreasingly used for developing various therapeutic agents.

The potential use of IL-12 as an effective vaccine or therapeutic agentfor various diseases requiring cellular immune responses, as mentionedabove, is also based on the hypothesis that IL-12 participates in theproliferation of memory Th1 cells and memory CTL (Stobie, L. et al.,Proc. Natl. Acad. Sci. USA, 97:8427-8432, 2000; Mortarini, R. et al.,Cancer Res., 60:3559-3568, 2000; Mbawuike, I. N. et al., J. Infect.Dis., 180:1477-1486, 1999). In particular, with respect to the mostsevere problems, metastasis and recurrence, upon treatment of varioustumors, the induction of memory immune responses is essential. However,to date, an accurate mechanism explaining these effects of IL-12 has notbeen known. Some recent reports suggest that, since increased levels ofIFN-γ during Th1 cell differentiation by IL-12 has an antiproliferativeeffect, IL-12 may induce memory immune responses by suppressingapoptosis of CD4⁺T cells (Fuss, I. J. et al., Gastroenterology117:1078-1088, 1999; Marth, T. et al., J. Immunol. 162:7233-7240, 1999).Also, another hypothesis involving IL-12 inducing memory 5 immuneresponses has been suggested, based on the notion that elevated levelsof IFN-γ by IL-12 promote expression of IL-15 participating in potentand selective stimulation of memory CD8⁺ T cells (Zhang, X. et al.,Immunity 8:591-599, 1998). These reports suggest that IL-12 mayparticipate in both primary immune responses and memory immuneresponses. Thus, IL-12 has a potential to be particularly valuably usedin vaccine immunization.

IL-12 as an adjuvant has been reported not to induce the uncontrolledproduction of other cytokines, not to induce any sensitization in thecase of originating from humans and to have no obvious side effects uponsubcutaneous injection.

When IL-12 is administered in a DNA form, its endogeneous expression isinduced, and the expression of IL-12 lasts for a longer period of timethan the case of being administered in a protein form. Based on thisfact, Sanjay Gurunathan et al. stated in Nature Medicine 4:1409-1415,1988 that the administration of an antigenic protein in combination withIL-12 DNA induces more long-lasting immune responses againstintracellular infections such as Leishmania major and Mycobacteriumtuberculosis.

Unlike these reports, the present inventors found that, when a proteinform of IL-12 used as an adjuvant is encapsulated in sustained releasemicrospheres and used in a vaccine composition, it sustains andremarkably enhances antibody and cellular immune responses to a vaccineeven in small amounts for a longer period of time than a DNA form ofIL-12.

In detail, the present inventors subcutaneously administered IL-12encapsulated in microspheres to mice in combination with a HBVpreventive vaccine, recombinant HBsAg. This combination resulted intotal IgG and IgG1 antibody responses 10 to 30-fold higher than HBsAgalone, HBsAg plus native form of IL-12 not encapsulated in microspheresand HBsAg plus IL-12 DNA. In particular, IgG2a antibody responses, as anindicator for Th1 immune responses, were found to remarkably increase by80 to 2000 times by the IL-12 encapsulated in microspheres. CTL immuneresponses were also found to increase about 6 times by the IL-12encapsulated in microspheres. In addition, when the IL-12 encapsulatedin microspheres was intranasally administered in combination with anM2/82-90 peptide of RSV, CTL responses were 5 to 10-fold elevated.Further, in an influenza HA vaccine model, the use of theIL-12-encapsulating microspheres induced 2 to 3-fold increased antibodyresponses and 4 to 25-fold increased CTL responses against aco-administered vaccine. These results indicate that theIL-12-encapsulating microspheres are applicable to various vaccines toenhance immune responses against the vaccines.

Thus, the IL-12, encapsulated in sustained release microspheres,contained in the vaccine composition of the present invention indicatesits protein form.

In comparison with a DNA form of IL-12, a protein form of IL-12,contained in the present vaccine composition as an adjuvant, has thefollowing advantages. Protein forms of cytokines are typicallyadministered to the body via the subcutaneous route, but subcutaneousinjection of cytokines in DNA forms is known to lead to unsatisfactoryeffects. In this regard, when a vaccine in a protein form isadministered subcutaneously while a DNA form of IL-12 as an adjuvant isadministered intramuscularly, the vaccine antigen and the adjuvant donot exist simultaneously in an identical region, thereby making itdifficult to attain desired effects. In addition, IL-12 should bepresent in the early phase of the antigen presentation to be served asan adjuvant for a co-administered vaccine. However, when theimmunization is carried out by intramuscularly administering IL-12 DNA,it takes much time for IL-12 DNA to express in the body (generallymuscular cells) and move to a desired site. In particular, the use of anIL-12 protein in a form of being encapsulated in microspheres make itpossible to control the in vivo release duration by varying thecomposition of the microspheres. In contrast, in the case of using IL-12DNA, IL-12 DNA expresses in very low levels, the persistence ofexpressed IL-12 is not controlled, and clinical safety is not ensured,thereby requiring further studies.

The term “IL-12”, as used herein, refers to an IL-12 protein, a subunitthereof, a multimer of the subunit, a functional fragment of IL-12, anda functional equivalent and/or isoform of IL-12. The functional fragmentof IL-12 includes fragments that induce immune responses to an antigenwhen administered together with the antigen. In addition, the functionalequivalent or isoform of IL-12 includes IL-12 variants that are alteredto have biological activity similar to native IL-12, that is, modifiedIL-12 proteins having an ability to induce an immune response to anantigen when administered together with the antigen. In particular, thisincludes modified IL-12 proteins with an alteration of a specific aminoacid residue, which are designed to have higher immunoenhancingactivity.

IL-12 may be obtained from various origins or synthesized using a knowntechnique. For example, IL-12 may be purified (isolated) from a nativeorigin (e.g., mammals such as humans), produced by chemical synthesis,or produced by a recombinant DNA technique. In addition, IL-12 may beobtained from commercially available products. In particular, IL-12 maybe preferably isolated, synthesized or produced by a recombinant DNAtechnique from a human origin.

IL-12 as an adjuvant may be used in an amount of about 1 ng to about 20μg, and preferably about 100 ng to about 5 μg, but the present inventionis not limited to this range.

A majority of proteins, when orally administered, lose their activestructures under the acidic environment of the stomach, are destroyed byenzymatic degradation, and are absorbed in very low levels by the mucousmembrane of the stomach and the intestinal. Thus, most protein drugs areadministered parenterally, that is, by intravenous injection,subcutaneous injection or intramuscular injection. Even afteradministration via these routes, most protein drugs should be repeatedlyinjected due to their short half-lives. For controlled release of theseproteins, these ingredients may be included in a dispersion systemselected from the group consisting of macromolecular complexes,nanocapsules, microspheres, beads, oil-in-water emulsions, micelles,mixed micelles, liposomes and resealed erythrocytes.

The most commonly used biodegradable polymers for sustained injectablepreparations of proteins are polyesters as synthetic polymers, whichinclude polylactide (PLA), polyglycolide (PGA) and their copolymer,poly(lactide-co-glycolide) (PLGA). In addition to these syntheticpolyesters, natural polymers are studied as matrices for sustainedformulations of protein drugs, which include lipids such as lipids,fatty acids, waxes and their derivatives; proteins such as albumin,gelatin, collagen and fibrin; and polysaccharides such as alginic acid,chitin, chitosan, dextran, hyaluronic acid and starch. Non-limitingexamples of the lipids include fatty acids (e.g., myristic acid,palmitic acid, stearic acid, etc.), monoacylglycerols (e.g., pamoicacid, glyceryl myristate, glyceryl palmitate, glyceryl stearate, etc.),sorbitan fatty acid esters (e.g., sorbitan myristate, sorbitanpalmitate, sorbitan stearate, etc.), triglycerides (e.g., diacylglycerol, trimyristin, tripalmitin, tristearin, etc.), phospholipids(e.g., phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylacid, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol,cardiolipin, etc.), sphingolipids (e.g., sphingosine, ceramide,sphinganine, etc.), waxes, and salts and derivatives thereof.

In particular, among the aforementioned biodegradable polymers, thepolyesters, such as PLA, PGA or PLGA, are approved to be biocompatibleand safe to the body because they are metabolized in vivo to harmlesslactic acid and glycolic acid by hydrolysis. The degradation of thepolyesters may be controlled at various rates according to the molecularweight, the ratio of the two monomers, the hydrophilicity, and the like,for various durations ranging from a short period of one to two weeks toa long period of one to two years. The polyesters are polymericsubstances that have been approved for use in humans in several tens ofcountries, including by the U.S. Food and Drug Administration (FDA), andcommercialized. Therefore, the polyesters may be preferably used in thepresent invention. In particular, the polyesters such as PLGA or PLA maybe preferably used in the present invention.

To capture a protein into the aforementioned polymeric matrix, variousmethods may be used, including coacervation, spray drying-dependentencapsulation, and solvent evaporation in an organic or water phase.Among the above methods, W/O/W double emulsion-solvent evaporation hasbeen widely used in manufacturing sustained release microparticlescontaining protein drugs because most protein drugs are water-soluble.In this W/O/W technique, a protein or water-soluble drug is dissolved inwater, and this aqueous phase is dispersed in an organic phasecontaining a biodegradable polymer using an ultrasonicator orhomogenizer, in order to give a primary emulsion. Again, this primaryemulsion is dispersed in a secondary aqueous phase containing asurfactant such as polyvinylalcohol, so as to provide a secondaryemulsion. As the organic solvent is removed from this system by heatingor under pressure, the polymer is solidified to form microparticles. Themicroparticles are recovered by centrifugation or filtration andfreeze-dried to give biodegradable microparticles containing the proteinor water-soluble drug.

To minimize denaturation and irreversible coagulation of a protein whenthe protein is entrapped into a biodegradable polymer, a stabilizer maybe used in an aqueous solution of the protein, which is exemplified bytrihalose, mannitol, dextran and polyethylene glycol. These stabilizersform a hydrated layer around a protein and thus reduce the interactionbetween a protein and an organic solvent, thereby preventing thedenaturation and irreversible coagulation of the protein to some extent.In addition, the protein denatruation may be minimized by directlydispersing in an organic solvent a protein drug in a powder form ratherthan in a form of being dissolved in an aqueous solution.

The term “sustained or controlled release”, as used herein, means thatthe vaccine composition of the present invention, containing an IL-12adjuvant encapsulated in microspheres, requires an hour or longer torelease a major portion of the active substance into the surroundingmedium, for example, 24 hours or longer.

Microsphere-based drugs may be utilized for oral ingestion,implantation, or external application to the skin or a mucous membrane.Where implantation is desired, microspheres may be implantedsubcutaneously, constitute a portion of a prosthesis, or be insertedinto a cavity of the human body. Subcutaneous implantation using asyringe consists of injecting an implant directly into a subcutaneoustissue, and is a particularly effective method for controlled drugdelivery. The IL-12-encapsulating microspheres according to the presentinvention may be suspended in a physiological buffer and introduced intoa desired site using a syringe.

When applied to a desired site of the body by a desirable mode, theIL-12-encapsulating sustained release microspheres provides sustainedrelease of IL-12 by allowing IL-12 to diffuse through the microspheresor by allowing the microspheres to degrade in vivo upon contact withbody fluids. When the microspheres are degraded in a site where themicrospheres are injected, the degree of their degradation, that is, therelease rate of the active substance, may be regulated by the degree ofcrosslinking of the microspheres.

The IL-12-encapsulating microspheres may be about 20 nm to 50 μm indiameter. The microspheres of this sphere size may be suspended in apharmaceutical buffer and introduced into a patient using a syringe.

The vaccine composition containing IL-12 encapsulated in microspheresaccording to the present invention may be administered to a patient,whether displaying a pathogenic state caused by a pathogen or not, so asto suppress or delay the incidence of a disease or alleviate oreliminate the disease.

The vaccine composition for prevention or therapy according to thepresent invention may be administered in an immunologically effectiveamount for prevention or therapy. The term “immunologically effectiveamount” means an amount suitable for inducing an immune response. Aspecific amount may vary depending on the patient's age and weight, theseverity of illness and administration methods, and a suitable amountmay be easily determined by those skilled in the art. The vaccinecomposition may be contained in a pharmaceutically or physiologicallyacceptable vehicle, for example, physiological or phosphate-bufferedsaline, or ethanol or polyols, such as glycerol or propylene glycol.

If desired, the vaccine composition of the present invention may furtherinclude additional adjuvants (e.g., vegetable oils or emulsionsthereof), surfactants (e.g., hexadecylamine, octadecyl amino acidesters, octadecylamine, lisolecithin, dimethyldioctadecylammoniumbromide, N,N-dioctadecyl-N′, N′-bis (2-hydroxyethylpropane diamine),methoxyhexadecylglycol, pluronic polyols), polyamines (e.g., pyrans,dextransulfate, poly IC, carbopol), peptides (e.g., dimethylglycine),immunostimulatory complexes, oil emulsions, lipopolysaccharides (e.g.,d3-MPL (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research,Inc., Hamilton, Mont.)), and inorganic gels.

The vaccine composition of the present invention may be administered byvarious routes, for example, parenterally, intraarterially,subcutaneously, transdermally, intramuscularly, intraperitoneally,intravenously, orally and intranasally.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

EXAMPLE 1 Preparation of rIL-12-Encapsulating Microspheres and MockMicrospheres

IL-12-encapsulating microspheres were prepared by a W/O/W doubleemulsion-solvent evaporation method.

A murine recombinant IL-12 protein (rIL-12) (R&D System) and bovineserum albumin (BSA) were added to PBS buffer according to thecomposition summarized in Table 1, below, so as to give a W1 solution(total volume: 500 μl). The W1 solution was emulsified in 1.2 ml of DCM(dichloromethane) (oil phase (O)) supplemented with a polymeric carrierPLGA (polylactide-co-glycolide) and an emulsifier Pluronic L121 using ahomogenizer, thus providing a primary emulsion (W1/O). Again, theprimary emulsion was emulsified in distilled water (W2) containinganother emulsifier PVA (polyvinylalchol) using a homogenizer, thusproviding a secondary emulsion (W1/O/W2). The secondary emulsion wassolidified to form microspheres, filtered and dried. TABLE 1 W1 Oil W2mlL-12 BSA Buffer PLGA CH₂Cl₂ 1% PVA 50 μg 12.5 mg 500 μg 500 mg 1.2 mlof 2% pluronic L121

The rIL-12-encapsulating microspheres were analyzed using a laserscattering particle size distribution analyzer (Hydro-2000MU, MALVERN)for sphere size, an optical microscope (IX70, Olympus) and a SEMmicroscope (JSM 890, JEOL LTD) for morphology, and a size exclusion(SE)-HPLC column (TOSOH) and a Dc protein analyzer (Bio-Rad) for loading(%).

Mock microspheres as a negative control were prepared according to thesame procedure as described above except for not using rIL-12.

EXAMPLE 2 Enhanced HBsAg-Specific Antibody Responses by therIL-12-Encapsulating Microspheres

The adjuvant effect of the rIL-12-encapsulating microspheres withrespect to antibody responses was investigated as follows. A hepatitis Bvirus surface antigen, HBsAg (Euvax B, LGCI Co. Ltd.) and themicrospheres prepared in Example 1 were suspended in 100 μl of asuspension solution (3% carboxymethyl celluose, 8.7 mg/ml NaCl, 0.1%Tween 20). Five-week old BALB/c CrSlc mice were subcutaneously immunizedwith the resulting suspension. After four weeks, the titers of totalserum IgG, IgG1, and IgG2a antibodies were measured by an anti-S ELISAto determine whether anti-HBsAg antibody responses had been induced. InFIGS. 1 a, 1 b, 1 c, 2 a, 2 b and 2 c, antibody responses were expressedas absorbance at 450 nm. FIGS. 1 d, 1 e and 1 f show the results ofquantitative comparison for antibody responses expressed as antibodytiters measured by an end-point dilution assay.

As shown in FIG. 1 a, the strongest total IgG antibody responses wereobserved in Group 4 administered with the rIL-12-encapsulatingmicrospheres. As shown in FIG. 1 d, the Group 4 was also found toproduce about 9 to 27-fold stronger total IgG antibody responses thanother groups. In contrast, in both Group 2 administered with mockmicrospheres and Group 3 administered with mock microspheres plus rIL-12protein, no significant increase was observed (see, FIGS. 1 a and 1 d).Also, in the case of IgG1 responses, the Group 4 administered with therIL-12-encapsulating microspheres was found to induce about 9-foldstronger immune responses (see, FIGS. 1 b and 1 e). In the case of IgG2aresponses, only the Group 4 administered with the rIL-12-encapsulatingmicrospheres induced very-strong significant antibody responses (see,FIG. 1 c). As shown in FIG. 1 f, the Group 4 was found to induce 81 to2187-fold stronger IgG2a antibody responses than other groups.

These results indicate that the rIL-12-encapsulating microspheresenhance host's antibody and T-helper 1 immune responses to aco-administered antigen, and that the present microspheres designed tocontinuously release IL-12 greatly improve the adjuvant effect of IL-12.

Also, mice were immunized with different amounts of the antigen, and theadjuvant effect of the microspheres was evaluated by anti-S ELISA. Asshown in FIGS. 2 a to 2 c, even when the antigen was used even in smallamounts, the co-administration of the IL-12-encapsulating microspheresalso was found to lead to strong antibody responses. These resultsindicate that the present microspheres have an excellent effect onadjuvantation of an antigen regardless of administered amounts of theantigen.

EXAMPLE 3 Enhanced HBsAg-Specific CTL Responses by therIL-12-Encapsulating Microspheres

The adjuvant effect of the rIL-12-encapsulating microspheres withrespect to CTL responses was investigated as follows. HBsAg (Euvax B,LGCI Co. Ltd.) and the microspheres were suspended in 100 μl of asuspension solution (3% carboxymethyl celluose, 8.7 mg/ml NaCl, 0.1%Tween 20). Five-week old BALB/c CrSlc mice were subcutaneously immunizedwith the resulting suspension. After 13 weeks (primary test) and after 9and 24 weeks (secondary test), the spleen was excised from the immunizedmice, and CD8⁺ T cells were isolated from the spleen by a magnetic beadcell separation technique (MACS). The isolated CD8⁺ T cells weresubjected to an IFN-γ ELISPOT assay using HBV S-specific CTL epitope(IPQSLDSWWTSL) as a stimulus.

FIG. 3 a shows the results 13 weeks after immunization. As shown in FIG.3 a, a group co-administered with the rIL-12-encapsulating microspheresdisplayed remarkably enhanced CTL responses in comparison with othergroups. As shown in FIGS. 3 b and 3 c, like the results of antibodyresponses, this excellent effect of the rIL-12-encapsulatingmicrospheres on enhancing CTL responses was found to be achievedregardless of the amount of the antigen used in the immunization. Inaddition, this enhancement of CTL responses by the rIL-12-encapsulatingmicrospheres was maintained 24 weeks after immunization (see, FIG. 3 c).

EXAMPLE 4 Enhanced RSV-Specific CTL Responses by the rIL-12Encapsulating Microspheres

To determine whether the rIL-12-encapsulating microspheres have thevaccine adjuvanting effect on another antigen, a respiratory syncytialvirus (RSV) was used as a vaccine antigen. In addition, therIL-12-encapsulating microspheres were evaluated for theirimmunoenhancing effects upon the use of an antigen of a peptide typeinstead of a protein type and upon the intranasal administration of themicrospheres instead of subcutaneous injection. First, an M2/82-90peptide (Peptron Co. Ltd.), identified as a CD8⁺ T cell epitope, and theIL-12-encapsulating microspheres were suspended in 50 μl of a suspensionsolution (PBS). Five-week old BALB/c CrSlc mice were intranasallyimmunized twice at intervals of 2 weeks with the resulting suspension.After two weeks, lung lymphocytes were isolated from the immunized mice,and FACS was carried out to determine whether RSV M2/82-90 specific CTLresponses are induced. FIG. 4 a shows the results of quantitativeanalysis using FACS for the percentage of M2/82-90-specific CD8⁺ T cellsamong total lung CD8⁺ T cells. FIG. 4 b shows the results ofquantitative analysis using FACS of stained cells for the percentage ofIFN-γ-positive M2/82-90-specific CTL. As shown in FIG. 4 a, incomparison with a mock microsphere-administered group, in arIL-12-encapsulating microsphere-administered group, M2/82-90-specificCD8+T cells were significantly increased. In addition, as shown in FIG.4 b, in the rIL-12-encapsulating microsphere-administered group,IFN-y-secreting M2/82-90-specific CTL was significantly increased incomparison-with the other group. These results indicate that therIL-12-encapsulating microspheres are applicable not only to the subunitvaccine but also to the peptide vaccine and applicable various types ofantigens regardless of the administration route of the microspheres.

EXAMPLE 5 Comparison of the rIL-12 Protein-Encapsulating Microspheresand IL-12 DNA for Adjuvant Effects

To compare a DNA form of an adjuvant vaccine, known to continuouslyinduce protein expression, and a protein form of the adjuvant,encapsulated in microspheres, for adjuvant effects, five-week old BALB/cCrSlc mice were subcutaneously immunized with HBsAg (Euvax B, LGCI Co.Ltd.) and the IL-12-encapsulating microspheres. After two weeks, thetiters of total serum IgG, IgG1, and IgG2a antibodies were measured byan anti-S ELISA. Separately, five-week old BALB/c CrSlc mice wereimmunized with HBsAg by subcutaneous injection and IL-12 DNA(ACP30-mIL-12, POSTECH Cellular Immunology Lab.) by intramuscularinjection, and, after two weeks, the titers of total serum IgG, IgG1,and IgG2a antibodies were measured by an anti-S ELISA. As shown in FIGS.5 a to 5 c, a rIL-12-encapsulating microsphere-administered group (Group3) was found to induce stronger HBsAg-specific total IgG, IgG1, andIgG2a antibody responses than an IL-12 DNA-administered group (Group 2).These results indicate that the rIL-12-encapsulating microspheres of thepresent invention are superior as an adjuvant to the IL-12 DNA known toinduce sustained expression of a gene encoding IL-12.

EXAMPLE 6 Enhanced Influenza HA-specific Antibody Responses by therIL-12-Encapsulating Microspheres

To investigate the adjuvant effect of the rIL-12-encapsulatingmicrospheres with respect to antibody responses, five-week old BALB/cCrSlc mice were intranasally immunized twice at intervals of two weekswith an influenza HA protein (Influenza HA vaccine, LG Household &Health Care Co. Ltd.) and the microspheres prepared in Example 1, whichboth were suspended in a suspension solution (3% carboxymethyl celluose,8.7 mg/ml NaCl, 0.1% Tween 20). After eight weeks, the titers of totalserum IgG, IgG1, and IgG2a antibodies were measured by an anti-HA ELISAto determine whether antigen-specific antibody responses had beeninduced. FIG. 6 shows the results of the quantitative comparison of testgroups for antibody responses by an end-point dilution assay. As shownin FIG. 6, Group 2, administered with the antigen and rIL-12, inducedalmost identical antibody responses to Group 4 administered withone-fifth of the amount of the rIL-12-encapsulating microspheres used inGroup 2. In contrast, in Group 3 administered with therIL-12-encapsulating microspheres in the same amount as in Group 2,total serum IgG, IgG1 and IgG2a antibody responses were significantlyincreased. In particular, with respect to IgG2a responses, Group 3,administered with the rIL-12-encapsulating microspheres, induced muchstronger antibody responses than other groups.

These results indicate that the rIL-12-encapsulating microsphereseffectively increase antigen-specific antibody responses and Th1 immuneresponses and are applicable diverse antigens other than HBsAg.

In addition, when Group 3 and Group 4, immunized with different amountsof the rIL-12-encapsulating microspheres, were compared with each other,antibody responses were increased along with the administered amount ofthe microspheres.

EXAMPLE 7 Enhanced Influenza HA-specific CTL Responses by therIL-12-Encapsulating Microspheres

To investigate the adjuvant effect of the rIL-12-encapsulatingmicrospheres with respect to CTL responses, five-week old BALB/c CrSlcmice were intranasally immunized twice at intervals of two weeks with aninfluenza HA protein (Influenza HA vaccine, LG Household & Health CareCo. Ltd.) and the microspheres prepared in Example 1, which both weresuspended in a suspension solution (3% carboxymethyl celluose, 8.7 mg/mlNaCl, 0.1% Tween 20). After 11 weeks, virus infection was carried outwith an influenza virus. Five days after the virus infection, lungs wereexcised from the mice, and lung lymphocytes were isolated by aLympho-prep technique. CD8⁺ T cells in the lung were isolated,stimulated with an influenza HA-specific CLT epitope, and stained withCD8+ and IFN-γ-spcific antibodies. IFN-γ-secreting HA-specific CD8+ Tcell levels were analyzed by FACS.

As shown in FIGS. 7 a to 7 d, Group 2, administered with rIL-12, had nosignificant difference with Group 1 in CTL responses. In contrast, Group3, administered with the rIL-12-encapsulating microspheres, induced muchstronger CTL responses than other groups.

With respect to immune responses by memory T cells produced afterimmunization of mice, these results indicate that therIL-12-encapsulating microspheres are effective in enhancing immuneresponses by antigen-specific memory T cells.

EXAMPLE 8 Improved Protection of Immunized Mice Against InfluenzaChallenge by the rIL-12-Encapsulating Microspheres

To determine whether enhanced antibody and CTL responses by rIL-12encapsulated microsphere is correlated with in vivo protection againsthomologous influenza challenge, five-week old BALB/c CrSlc mice wereintranasally immunized twice at intervals of two weeks with an influenzaHA protein (Influenza HA vaccine, LG Household & Health Care Co. Ltd.)and the microspheres, which both were suspended in a suspension solution(3% carboxymethyl celluose, 8.7 mg/ml NaCl, 0.1% Tween 20). After 11weeks, the vaccinated mice were challenged with lethal doses ofinfluenza virus. As shown in FIG. 8, in which mice were compared betweentest groups for survival rate for nine days after the virus challenge,Group 2 administered with rIL-12 displayed a slightly increasedviability of about 10%, which was not significant, in comparison with acontrol group, Group 1, not administered with the adjuvant. In contrast,Group 3, administered with the rIL-12-encapsulating microspheres,exhibited a significantly increased viability of about 65%.

These results indicate that the rIL-12-encapsulating microspheres alsoeffectively increase host's protection against infectious diseases bysignificantly increasing antigen-specific antibody responses and CTLresponses.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the present invention provides a vaccinecomposition comprising a pathogenic antigen and an IL-12 adjuvantencapsulated in sustained release microspheres. IL-12, as an adjuvant inthe vaccine composition, is released in vivo for a prolonged period oftime by being encapsulated in sustained release microspheres, therebymaximizing its adjuvant effect.

1. A vaccine composition for enhancing an adjuvant effect of IL-12comprising a pathogenic antigen and an IL-12 adjuvant encapsulated incontrolled release microspheres.
 2. The vaccine composition as set forthin claim 1, wherein the pathogenic antigen is selected from the groupconsisting of viruses, bacteria, parasites and fungi.
 3. The vaccinecomposition as set forth in claim 2, wherein the pathogenic antigen isselected from the group consisting of hepatitis B virus, hepatitis Cvirus, human immunodeficiency virus, influenza virus and mycobacteria.4. The vaccine composition as set forth in claim 1, wherein thepathogenic antigen is in a protein or peptide form.
 5. The vaccinecomposition as set forth in claim 1, wherein the IL-12 is a recombinantIL-12.
 6. The vaccine composition as set forth in claim 1, wherein thecontrolled release microspheres are manufactured by doubleemulsion-solvent evaporation.
 7. A method of enhancing an adjuvanteffect of IL-12, which is characterized by employing IL-12 encapsulatedin controlled release microspheres as an adjuvant in a vaccinecomposition comprising a pathogenic antigen.
 8. The method as set forthin claim 7, wherein the vaccine composition is administeredsubcutaneously or intranasally.